This application claims priority to and benefit of provisional application USSN 60/129,009, filed Apr. 13, 1999, pursuant to 35 USC 119 (e).
Pursuant to 37 C.F.R. 1.71 (e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
The invention relates to methods and compositions for generating, modifying, adapting, and optimizing polynucleotide sequences that encode proteins having enzyme activities involved in starch metabolism which are useful for introduction into plant species, and other hosts, and related aspects.
Genetic engineering of agricultural organisms dates back thousands of years to the dawn of agriculture. The hand of man has selected the agricultural organisms having the phenotypic traits that were deemed desirable, e.g., taste, high yield, caloric value, ease of propagation, resistance to pests and disease, and appearance. Classical breeding methods to select for germplasm encoding desirable agricultural traits had been a standard practice of the world""s farmers long before Gregor Mendel and others identified the basic rules of segregation and selection. For the most part, the fundamental process underlying the generation and selection of desired traits was the natural mutation frequency and recombination rates of the organisms, which are quite slow compared to the human lifespan and make it difficult to use conventional methods of breeding to rapidly obtain or optimize desired traits in an organism.
The recent advent of non-classical, or xe2x80x9crecombinantxe2x80x9d genetic engineering techniques has provided new means to expedite the generation of agricultural organisms having desired traits that provide an economic, ecological, nutritional, or aesthetic benefit. To date, most recombinant approaches have involved transferring a novel or modified gene into the germline of an organism to effect its expression or to inhibit the expression of the endogenous homologue gene in the organism""s native genome. However, the currently used recombinant techniques are generally unsuited for substantially increasing the rate at which a novel or improved phenotypic trait can be evolved. Essentially all recombinant genes in use today for agriculture are obtained from the germplasm of existing plant and microbial specimens, which have naturally evolved coordinately with constraints related to other aspects of the organism""s evolution and typically are not optimized for the desired phenotype(s). The sequence diversity available is limited by the natural genetic variability within the existing specimen gene pool, although crude mutagenic approaches have been used to add to the natural variability in the gene pool.
Unfortunately, the induction of mutations to generate diversity often requires chemical mutagenesis, radiation mutagenesis, tissue culture techniques, or mutagenic genetic stocks. These methods provide means for increasing genetic variability in the desired genes, but frequently produce deleterious mutations in many other genes. These other traits may be removed, in some instances, by further genetic manipulation (e.g., backcrossing), but such work is generally both expensive and time consuming. For example, in the flower business, the properties of stem strength and length, disease resistance and maintaining quality are important, but often initially compromised in the mutagenesis process.
STARCH METABOLISM IN PLANTS
The biosynthesis of starches in higher plants occurs in three steps, the first of which involves synthesis of ADP-glucose from ATP and glucose-l-phosphate and is catalyzed by ADP-glucose pyrophosphorylase (xe2x80x9cADPGPPxe2x80x9d; EC 2.4.7.27). The second step of starch biosynthesis is transfer of a glucosyl moiety of ADP-glucose to a maltodextrin or starch to give rise to a new 1,4-glucosyl linkage; the reaction is catalyzed by a starch synthase (xe2x80x9cSSxe2x80x9d; EC 2.4.1.21), of which there are several forms present either as soluble enzymes or bound to starch particles as particulate enzymes. The third reaction is catalyzed by branching enzymes (xe2x80x9cBExe2x80x9d; EC 2.4.1.18) and is responsible for synthesis of 1,6-glucosyl linkages. An exemplary starch biosynthetic pathway is illustrated in FIG. 1.
Starch metabolism is a dynamic process wherein catabolic activities antagonize the synthetic (anabolic) processes which form starch. Examples of catabolic activities include amylase (alpha and beta), two categories of debranching enzymes, isoamylases and pullulanases (limit dextrinases; R enzymes), and starch phosphorylase. The composition of starches that are produced result from the relative actions of the anabolic and catabolic activities.
The enzymatic activities of the various enzymes involved in starch metabolism control the properties and types of the starches which are present in the plant, typically in the form of storage granules. Various commercial native starches produced in a variety of plants differ dramatically in important physical, mechanical, and chemical properties, and are important for foodstuff and industrial uses (Swinkels, J.(1985) Starch 37:1). It is theoretically possible to alter the composition of starches made in a plant cell or plant storage organ by introducing heterologous or modified genes encoding enzymes that can alter starch metabolism. U.S. Pat. Nos. 5,750,875 and 5,824,790 disclose methods that reportedly modify starch metabolizing ability by introducing foreign genes into a plant genome or by suppressing endogenous gene expression. However, both of these methods are severely limited by the small pool of naturally occurring genes in various organisms that are useful for the methods. It would be highly desirable for the art to have methods for producing novel starch compositions by engineering gene sequences encoding modified starch biosynthetic enzymes, and introducing these gene sequences into plant cells, thereby creating novel plant cells that produce a desirable starch composition, particularly of types which are industrially useful and not available or obtainable only by laborious purification and chemical modification methods.
As noted, the advent of recombinant DNA technology has provided agriculturists with additional means of modifying plant genomes. While certainly practical in some areas, to date genetic engineering methods have had limited success in transferring or modifying important biosynthetic or other pathways, including certain naturally-occurring genes encoding starch metabolizing enzymes into photosynthetic organisms and bacteria. The creation of plants and other photosynthetic organisms having improved starch biosynthetic pathways can provide increased yields of certain types of starchy foodstuffs, enhanced industrial feedstocks, improved chemical compositions and clothing, and may alter the types and properties of polyglucan polymers available for a wide range of industrial and pharmaceutical uses, among other desirable phenotypes.
Thus, there exists a need for improved methods for producing plants and agricultural photosynthetic microbes comprising heterologous gene sequences which encode one or more enzyme(s) that result in production of an improved starch composition. In particular, these methods should provide general means for producing novel starch metabolic enzymes, including increasing the diversity of the starch metabolic enzyme gene pool and the rate at which genetic sequences encoding one or more starch metabolic enzyme species having desired properties are evolved. It is particularly desirable to have methods which are suitable for rapid evolution of genetic sequences to function in one or more plant species and confer an improved starch phenotype (e.g., increased control over branching structures, improved physiochemical properties, improved yield, enhanced cross-linkability, incorporation of advantageous moieties, improved catalytic efficiency via increasing Vmax and/or increasing the apparent affinity of substrates for a starch metabolizing enzyme, and/or as a source of purifiable enzymes for in vitro starch synthesis and modification, as well as plants which express the novel genetic sequence(s), and the uses of said plants and starch compositions.
The present invention meets these and other needs and provides such improvements and opportunities. The disclosed method for providing an agricultural organism having an improved NSME enzymatic phenotype by iterative gene shuffling and phenotype selection is a pioneering method which enables a broad range of novel and advantageous agricultural compositions, methods, kits, uses, plant cultivars, and apparatus which will be apparent to those skilled in the art in view of the present disclosure. Other features and advantages of the invention will be apparent from the following description of the drawings, preferred embodiments of the invention, the examples, and the claims.
In a broad aspect, the invention provides a method for obtaining a polynucleotide encoding a novel protein, having a unique or improved property, that can participate in starch metabolism, either catabolically or anabolically. Such a novel protein is called generically a Novel Starch Metabolic Enzyme (xe2x80x9cNSMExe2x80x9d). The NSME generally has one or more of the following enzymatic activities: starch synthase (starch synthase), amylase (alpha or beta type), branching enzyme (BE, BEI, BEIIa, BEIIb, BEIII, and the like), debranching enzyme (isoamylase or pullulanase), starch phosphorylase, or modified activities thereof. The method involves the following steps: (1) sequence shuffling of a plurality of polynucleotide species having sequence similarity to one or more naturally occurring genes encoding a plant, animal, or microbial enzyme involved in starch metabolism, thereby forming a library of recombinant or xe2x80x9cshufflantxe2x80x9d sequences, (2) expressing the shufflant sequences in a population of host cells or organisms, such that each species of shufflant sequence is expressed in a discrete host cell or organism (or its progeny), and (3) selecting those host cells or organisms which express a desirable starch metabolic phenotype. Usually, the desirable starch metabolic phenotype is conferred by a modified enzyme activity of a starch metabolizing detected by any suitable method, such as enzyme assay, analysis of produced starch(es), and the like. Typically, the shufflant polynucleotides are recovered from the selected host cells or organisms and subjected to at least one additional round of sequence shuffling. Often, the process of shuffling, expression, selection, and recovery are performed recursively until the shuffling process yields sequences encoding proteins that have the desired starch metabolic phenotype. The resultant selected sequence(s) encode novel proteins having desired enzymatic activities in starch metabolism, and can be transferred in expressible form into plant cells, plants, or microbial cells participating in starch synthesis to yield novel starch compositions having desirable properties or chemistries.
In an embodiment, the invention provides an improved starch synthase or glycogen synthase, or shufflant thereof, and a polynucleotide encoding the same. In some embodiments, the polynucleotide is operably linked to a transcription regulation sequence forming an expression construct, which can be linked to a selectable marker gene; for embodiments where it is necessary to target a bacterial glycogen synthase shufflant into plant cell plastids, a sequence encoding a chloroplast transit peptide (CTP), such as that derived from Arabidopsis rbcS gene or amyloplast transit peptide, such as is known in the art, is fused in-frame to the shufflant glycogen synthase sequence, to ensure delivery of the glycogen synthase to the plastid compartment. In some embodiments, such a polynucleotide is present as an integrated transgene in a plant chromosome in a format for expression and processing of the glycogen synthase. It can be desirable for such a polynucleotide transgene to be transmissible via germline transmission in a plant; in the case of bacterial gene sequences transferred to plant or algal cells, it is often accompanied by a selectable marker gene which affords a means to select for progeny which retain the transferred shuffled glycogen synthase gene sequence. In some embodiments, the transferred shuffled glycogen synthase gene sequence is derived by shuffling a pool of parental sequences, at least one of which encodes a bacterial glycogen synthase or a substantial portion thereof. Often, the transcription control sequences comprise tuber-specific or seed-specific promoters to overcome possible detrimental effects of constitutive expression; the same may be used for expressing other NSME encoding sequences.
In alternative embodiments, the invention provides NSMEs that comprise branching enzymes, amylases, debranching enzymes, starch posphorylases, or the like. Methods for generating and isolating novel shuffled polynucleotides encoding polypeptides having modified catalytic activity as one or more of a starch branching enzyme, an amylase, a debranching enzyme or a starch posphorylase, wherein the modified catalytic activity is altered by at least one-half log unit as compared to the protein encoded by the known naturally-occurring gene sequence having the highest percentage of sequence identity to the protein encoded by the shuffled polynucleotide. The method involves transferring a library of shuffled polynucleotides encoding a starch metabolizing enzyme into a population of host cells, thereby producing a population of transformed host cells. In preferred embodiments, the host cells are lacking in an endogenous starch metabolizing enzyme corresponding to one or more starch metabolizing enzyme encoded by the shuffled polynucleotides. A subpopulation exhibiting a desired starch metabolic phenotype is selected from the population of transformed host cells, thereby forming a selected subpopulation of host cells harboring selected shuffled polynucleotides. Typically, the selected shuffled polynucleotides are then recovered, and at least one subsequent round of sequence shuffling, transfer and selection is performed, until a selected shufflant encoding an NSME having a desired enzymatic phenotype is obtained.
The present invention provides expression polynucleotides, e.g., plant transgenes, encoding an NSME polypeptide operably linked to a transcription regulatory sequence functional in plant cells, and optionally to a plastid transit peptide encoding sequence. In preferred embodiments, the transcription regulatory sequence controls expression in the starch-storing tissues and organs of an adult plant, such as may be obtained from a transgenic regenerable plant cell harboring said transgene.
The invention provides plant cells, regenerated plants, transgenic plants, cultivars, seeds, cuttings, reprodutive organs, vegetative tissues, germplasm, isolated DNA, isolated nuclei, and the like, as well as algal cells and bacterial cells comprising expressible polynucleotides encoding an NSME, e.g., plants harboring an NSME transgene or transient expression construct. Often, such adult plants and plant tissues are obtained from regenerable plant cells transfected with the transgene; however, alternative means of introducing NSME-encoding sequences can be employed, including contacting plant tissues, seeds, cuttings, or whole plants with a solution containing the NSME sequences, either as transgenes, transient expression constructs, Agrobacterium tumefaciens vectors, or plant viral vectors and the like. In some variations, only the starch-storing organs of a plant are exposed to the means of introduction of NSME encoding sequences. In some variations, the plants are hybrids or other sterile variety incapable of sexual and/or asexual reproduction.
The invention provides the uses of: NSME polynucleotides; encoded NSME polypeptides; plants (including seeds) containing same; plant, algal, and bacterial cells expressing NSME polynucleotides; and starches produced by plants or cells expressing NSME polynucleotides.
The invention also provides compositions comprising harvested starch-storing organs of plants expressing an NSME encoding sequence and containing starch compositions which do not occur in nature or in the absence of expression of the NSME encoding sequence(s).
The invention also provides novel starch compositions produced by NSME-expressing cells and plants, said novel starch compositions having at least one chemical or physical property which is detectably distinctive from naturally-occurring starch compositions obtained from naturally-occurring cells and/or plants of the same species and grown under similar conditions. Such novel starch compositions are produced by cultivating starch-producing plant cells, plants, yeast, algae, or bacteria that harbor at least one expressible NSME gene; typically the NSME gene is a shuffled and selected starch synthase, glycogen synthase, ADP glucose pyrophosphorlyase, branching enzyme, debranching enzyme, amylase, starch phosphorylase, or the like.
As with many polymer-producing processes, the compositions produced thereby are complex compositions which are best described by reference to the specific process used to make the complex compositions. Thus, the invention provides a starch composition made by a plant cell, yeast cell, algal cell, or bacterium expressing at least one NSME gene that functions in starch synthesis. Various parameters of starch composition can be altered, including, but not limited to: glucosamine-enriched starches, mean main chain length, degree and mean length of branching, melting point, refraction index, tensile strength, viscosity, swelling volume, fractional lipid content, gelation, solubility, phosphate content, and other parameters known to those skilled in the art.
In an embodiment, the invention provides a method for producing starches having an enhanced proportion of derivatized or reactive sugars, the method comprising: incubating a NSME-expressing host cell in a medium containing a derivatized saccharide which can be incorporated into oligosaccharide by the NSME. Examples of derivatized saccahrides include UDP-glucosamine, ADP-glucosamine, UDP-glucose-6-amine, and the like.
In another embodiment, the invention provides a yeast host cell expressing at least one NSME encoded by a shufflant polynucleotide, wherein the NSME catalyzes at least one step in the incorporation of UDP-glucose-6-amine into starch.
The invention provides a novel method for assaying the composition of starches in a high-throughput screening assay, particularly for assaying the degree of branching of synthesized starches, the method comprising employing a mass spectroscope to identify fragmented portions of starches obtained from sample cells, such as from reaction vessels containing cells harboring a discrete species of NSME shufflant enzyme. Such starch composition assays can be used for a variety of uses, including screening for NSME shufflant host cells which produce oligosaccharides (starches) having a desired composition.
In one embodiment, the mass spectroscopy (MS) starch composition assay involves: obtaining starch samples from a plurality of host cells or host plants harboring expressible NSME shufflant polynucleotides and cultured in individual culture vessels; and subjecting the starch samples individually to mass spectroscopic analysis on a triple quadrupole mass spectrometer and employing tandem mass spectroscopic analysis, thereby determining the composition of each starch sample.
The invention further provides a kit for obtaining a polynucleotide encoding a NSME protein, or subunit thereof, having a predetermined enzymatic phenotype, the kit comprising a cell line suitable for forming transformable host cells and a collection sequence-shuffled polynucleotides formed by in vitro sequence shuffling. The kit often further comprises a transformation enhancing agent (e.g., lipofection agent, PEG, etc.) and/or a transformation device (e.g., a biolistics gene gun) and/or a plant viral vector which can infect plant cells or protoplasts thereof.